NADH dehydrogenase (NADH:ubiquinone oxidoreductase, NADH-D) is the first multienzyme complex (Complex I) in a chain of three complexes that make up the mitochondrial electron transport chain. The mitochondrial electron transport chain is responsible for the transport of electrons from NADH to oxygen and the coupling of this oxidation to the synthesis of ATP (oxidative phosphorylation) which provides the energy source for driving a cell's many energy-requiring reactions. NADH-D accomplishes the first step in this process by accepting electrons from NADH and passing them through a flavin molecule to ubiquinone, which transfers the electrons to the second enzyme complex in the chain.
NADH-D and the other members of the electron transport chain are located in the mitochondrial membrane. NADH-D is the largest of the three complexes with an estimated mass of 800 kDa comprising some 40 polypeptide subunits of widely varying size and composition. The polypeptide composition of NADH-D in a variety of mammalian species including rat, rabbit, cow, and man is very similar (Cleeter, M. W. J. and Ragan, C. I. (1985) Biochem. J. 230: 739-746). The best characterized NADH-D is from bovine heart mitochondria and is composed of 41 polypeptides (Walker, J. E. et al. (1992) J. Mol. Biol. 226: 1051-1072). Seven of these polypeptides are encoded by mitochondrial DNA while the remaining 34 are nuclear gene products that are imported into the mitochondria. Many of these imported polypeptides are characterized by various N-terminal peptide sequences that target them to the mitochondria and are then cleaved from the mature protein. A second group of polypeptides have neither N-terminal targeting sequences nor modified-N terminal amino acids. The import signals of this second group appear to be lie within the mature protein (Walker et al., supra).
The functions of many of the individual subunits in NADH-D are largely unknown. The 24-, 51-, and 75-kDa subunits have been identified as being catalytically important in electron transport, with the 51-kDa subunit forming part of the NADH binding site and containing the flavin moiety that is the initial electron acceptor (Ali, S. T. et al. (1993) Genomics 18:435-39). The location of other functionally important groups, such as the electron-carrying iron-sulfate centers, remains to be determined. Many of the smaller subunits (&lt;30 kDa) contain hydrophobic sequences that may be folded into membrane spanning .alpha.-helices. These subunits presumably are anchored into the inner membrane of the mitochondria and interact via more hydrophilic parts of their sequence with globular proteins in the large extrinsic domain of NADH-D.
The 49 kDa subunit of bovine NADH-D is associated with the iron-sulfate portion of the complex (Feamley, I. M. et al. (1989) EMBO J. 8:665-72). Although the 49 kDa subunit is a nuclear encoded polypeptide that is isolated together with a 30 kDa and a 13 kDa subunit, the function of the 49 kDa subunit is not known. The three proteins (13-, 30-, and 49-kDa) contain two iron-sulfur centers among them. However, no cysteine-containing motifs typical of iron-sulfate centers are found in the 49 kDa subunit, so there is no evidence that it contains one of the iron-sulfur centers. The 49 kDa subunit may be a transmembrane protein but it contains only one hydrophobic region of sufficient length to span the typical membrane (Fearnley et al. supra).
The MLRQ subunit is one of the small (9 kDa) subunits that is nuclear encoded and contains no signal peptide (Walker et al. supra). A potential membrane-spanning .alpha.-helix presumably anchors the MLRQ subunit to the inner membrane of the midtochondria, but the precise function of the subunit is unknown.
Defects and altered expression of NADH-D are associated with a variety of disease conditions in man, including neurodegenerative diseases, myopathies, and cancer (Singer, T. P. et al. (1995) Biochim. Biophys. Acta 1271:211-19; Selvanayagam, P. and Rajaraman, S. (1996) Lab. Invest. 74:592-99). In addition, NADH-D reduction of the quinone moiety in chemotherapeutic agents such as doxorubicin is believed to contribute to the antitumor activity and/or mutagenicity of these drugs (Akman, S. A. et al. (1992) Biochemistry 31:3500-6).
The discovery of new NADH-D subunits and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention and treatment of cancer and immune and smooth muscle disorders.